6. PROJECT SUMMARY/ABSTRACT Oxidative stress-induced intestinal epithelial barrier dysfunction has been implicated in the pathogenesis of a wide spectrum of intestinal inflammatory diseases including necrotizing enterocolitis, inflammatory bowel disease and infectious enterocolitis. A significant body of evidence indicates that barrier function of GI tract is disrupted in various GI diseases. Our studies during the past several years have demonstrated that oxidative stress disrupts intestinal epithelial tight junction by phosphorylation of occludin on tyrosine residues and dephosphorylation on threonine residues;these processes involve the intracellular signaling molecules such as c-Src, FAK, PI3K, PP2A, PKCh, PKCz and PP1. Our studies also demonstrated that EGF, a GI mucosal protective factor, prevents the oxidative stress-induced disruption of tight junctions;this process involved signaling molecules, PLCg, PKCbI, PKCe and MAP kinases. Our preliminary data have identified two tyrosine residues and two threonine residues in occludin that lie in a highly conserved motif of occludin and suggested that phosphorylation of these residues regulates the integrity of tight junctions. Therefore, we plan to further continue our effort to understand the regulation of phosphorylation of these amino acids in the oxidative stress- induced disruption of intestinal epithelial barrier function and EGF-mediated prevention of oxidative stress effects. On the basis of our results it is hypothesized that: a) c-Src-mediated phosphorylation of occludin on Y398 and Y402 mediates the H2O2-induced destabilization of intestinal epithelial tight junctions, b) H2O2- induced disruption of tight junctions involves of dephosphorylation of occludin on T403 and T404 by down regulation of PKCh, and c) EGF and probiotics prevent oxidative stress-induced tight junction disruption by a MAP kinase-dependent mechanism. We propose to determine that: 1) c-Src directly interacts with occludin and phosphorylates Y398/Y402 in H2O2-treated cells. 2) H2O2-induced Tyr-phosphorylation of claudins plays a role in the disruption of tight junctions. 3) c-Src plays a role in H2O2-induced attenuation of steady state dynamics of tight junction proteins. 4) H2O2 accelerates cell spreading and migration by c-Src-mediated phosphorylation of occludin, claudins. 5) PKCz regulates tight junction integrity by Thr-phosphorylation of tight junction proteins. 6) Oxidative stress disrupts tight junctions by down regulation of PKCh. 7) Dephosphorylation of tight junction proteins on Ser residues regulates tight junction integrity. 8) Phosphorylation of tight junction proteins on specific Ser/Thr residues regulates their self-association and actin binding affinity. 9) Probiotics protect tight junctions from H2O2 by inducing EGF receptor transactivation. 10) EGF and probiotics ameliorate ischemia/reperfusion-induced tight junction disruption in mouse intestine. 11) EGF and probiotics attenuate the H2O2-induced PKCh down regulation, alteration of occludin phosphorylation and tight junction disruption in human colonic biopsies. The outcome of these studies has a direct relevance to our understanding of the pathogenesis of many GI diseases and has the potential to contribute to the future development of new therapeutic strategies.